Alternative polyadenylation as a genetic regulatory mechanism to bridge genome to phenome in the nervous system

项目总结 神经性厌食症是最致命的心理疾病，据估计终生疾病死亡率为10%。 而超过三分之一的癌症患者会死于基于疾病的厌食症，而不是癌症本身。论 另一方面，超过三分之二的美国成年人超重或肥胖，这个数字以及 心脏病、糖尿病和癌症等相关合并症的发生率预计只会在 接下来的几年。尽管这些饮食失调的方向相反，但肥胖的饮食功能障碍 而厌食症是由中枢神经系统(CNS)中常见的食欲回路介导的。无数的研究 已经记录了这些食欲中多个基因产物的协调和复杂的变化模式 在暴饮暴食或暴饮暴食后的一段时间内会出现这些症状。这些观察结果强烈地表明， 饮食过量或不足的行为决定很可能是由多靶点、适应不良的基因驱动的 中枢神经系统食欲中心的重新编程过程。因此，一个核心问题是什么样的全球进程可以协调。 在多个基因产品中发生这样的变化？我发表的研究表明，食欲的变化与 伴随着下丘脑交替多聚腺苷(APA)的变化。APA是一种快速的、活性依赖的RNA 调节信使核糖核酸转录的稳定性、成熟度和定位的加工机制。我发现了一个 金属蛋白酶组织抑制因子2(Timp2)的APA图谱显著改变 在肥胖表型的发展过程中。因此，我正在探索这样一种假设，即Timp2 APA在弓状体内 下丘脑的核(ARC)测量肥胖的发展。我建议在F99上进行的实验 这一阶段将表明：1)Timp2 mRNA对ARC的食欲控制是必需的；2)APA对ARC的食欲控制是必需的 Arc Timp2是对抗吞噬和肥胖所必需的。这些研究将是第一个从功能上 将APA调控与取食行为联系起来，将成为进一步研究基因组与行为现象的基础 在我独立的职业生涯中学习。我的赞助人加里·韦曼博士和共同赞助人苏珊娜·阿普尔雅德博士 和艾米丽·夸尔斯-克里克莫尔，都是华盛顿州立大学的资深神经科学家，在 分子神经科学(Wayman)和摄食行为(Appleyard和Qualls-Creekmore)。我的建议 研究和培训计划将加强我对神经遗传学的理论和技术理解。在 博士前F99阶段，我将学习shRNA和CRISPR/SaCas9载体设计和验证策略，cell 培养技术、立体定向手术和先进的代谢分析。在博士后K00阶段，我 将在这些技能的基础上，学习使用遗传小鼠模型，多组学，先进的生物信息学，以及 人工智能计算模型将基因组映射到表现组调控。总体而言，拟议的培训将是最佳的 让我在一家领先的神经科学研究所开始独立的研究生涯，并推动 我们对RNA调控作为基因组和行为表现组之间的功能联系的理解。

PROJECT SUMMARY Anorexia nervosa is the deadliest psychological disorder with an estimated 10% lifetime disease mortality rate, while over 1/3 of all cancer patients will die from disease-based anorexia, not the cancer itself. On the opposite end of the spectrum, over 2/3 of US adults are overweight or obese, and this number, as well as the rates of associated comorbidities such as heart disease, diabetes, and cancer, is only expected to increase in the coming years. Despite the opposite directionality of these eating disorders, dysfunctional eating in obesity and anorexia is mediated by common appetite circuitry in the central nervous system (CNS). Numerous studies have documented a coordinated and complex pattern of changes in multiple gene products in these appetite centers following periods of excessive or inadequate eating. These observations strongly suggest that the behavioral decision to eat excessively or inadequately is likely driven by a multitargeted, maladaptive genetic reprogramming process in CNS appetite centers. Thus, a core question is what global process could coordinate such changes in multiple gene products? My published studies have demonstrated that appetite changes align with changes in alternative polyadenylation (APA) in the hypothalamus. APA is a rapid, activity-dependent RNA processing mechanism that regulates mRNA transcript stability, maturation, and localization. I identified a significant APA pattern change on tissue inhibitor of metalloproteinases 2 (Timp2), a gene previously implicated in the development of an obese phenotype. Thus, I am exploring the hypothesis that Timp2 APA in the arcuate nucleus (ARC) of the hypothalamus meters the development of obesity. My proposed experiments in the F99 phase will show that 1) Timp2 mRNA is necessary for appetite control in the ARC, and that 2) APA regulation of ARC Timp2 is necessary to counteract hyperphagia and obesity. These studies will be the first to functionally link APA regulation to feeding behavior and will serve as the basis of further genome to behavioral phenome studies in my independent career. My Sponsor, Dr. Gary Wayman, and Co-Sponsors, Drs. Suzanne Appleyard and Emily Qualls-Creekmore, are established neuroscientists at Washington State University with expertise in molecular neuroscience (Wayman) and ingestive behavior (Appleyard and Qualls-Creekmore). My proposed Research and Training plan will strengthen my theoretical and technical understanding of neurogenetics. In the pre-doctoral F99 phase, I will learn shRNA and CRISPR/SaCas9 vector design and validation strategies, cell culture techniques, stereotaxic surgeries, and advanced metabolic analyses. In the postdoctoral K00 phase, I will build upon these skills and learn to use genetic mouse models, multi-omics, advanced bioinformatics, and AI computational models to map genome to phenome regulation. Overall, the proposed training will optimally position me to start an independent research career at a leading neuroscience research institute and advance our understanding of RNA regulation as a functional link between the genome and the behavioral phenome.